Project Summary/ Abstract Background and Preliminary data: For KRAS mutant NSCLC, which represent 25-30% of lung adenocarcinomas, no approved targeted therapies exist. Standard first-line therapy for KRAS mutant NSCLC includes anti-PD-1/PD-L1 immune checkpoint inhibitors (ICIs) and chemotherapy. Despite recent data showing durable long-term responses of subsets of patients to ICIs, not all patients benefit from this therapy. In particular, patient bearing co-occurring mutations in STK11/LKB1 (Liver Kinase B1), which is inactivated in up to 30% of KRAS mutant lung cancers, respond poorly to ICIs. Thus, there remains a critical need to develop new therapies for this subset of patients. The discovery of direct covalent inhibitors of the KRASG12C mutant protein have reignited hopes that an effective KRAS targeted therapy may be within reach. The initial data presented from the phase I trial of AMG 510, the first KRASG12C inhibitor to enter the clinic, showed 5 of 10 NSCLC patients achieved partial responses. It will be crucial to determine biomarkers that predict response to KRASG12C inhibition and improve on this therapy for distinct subsets of patients. We recently showed that MAPK inhibition by MEK inhibitors combined with the potent and selective MCL-1 inhibitor AMG 176 resulted in apoptosis and tumor regression in a subset of KRAS NSCLC preclinical models. In follow-up studies, we have now observed that cell lines with the most sensitivity to this combination harbor co-occurring LKB1 mutations. Restoring LKB1 expression in LKB1-/- cell lines hampers apoptotic effect of MEK + MCL-1 inhibition in cell line model and xenograft mouse model. Knocking out LKB1 in WT cell lines restore the sensitivity towards MEK + MCL-1 inhibition. We showed in our mechanistic study that trametinib induces preferential sequestration of BIM by MCL-1 in KRAS-LKB1 models. Proposed plan: We will identify the molecular mechanism of LKB1-mediated sensitivity of KRAS mutant NSCLCs to MAPK + MCL-1 inhibition by integrate CRISPR-based genomic screens and mass spectrometry proteomics. In addition, we will define the apoptotic dependencies of KRAS mutant NSCLC patients with WT or mutant LKB1. Finally, we will rigorously the efficacy of combined KRASG12C + MCL-1 inhibition in KRAS-LKB1 NSCLC models. Significance: Our proposed experiments integrate detailed mechanistic studies with analysis of clinical samples and will lay pre-clinical foundation for the clinical investigation of combined KRASG12C + MCL-1 inhibitors (both are currently under clinical development). If successful, these studies will have the potential in directly leading to a clinical trial for KRAS-LKB1 patient that have poor therapeutic options and establish LKB1 as a genetic biomarker for guiding the selection of BH3 mimetics in combination therapies targeting KRAS mutant NSCLC. In addition, our results will reveal fundamental insights into a novel role for LKB1 in the regulation of mitochondrial apoptosis and BCL-2 protein network.